This is the third year of operation of the Systems Neuroscience Imaging Resource (SNIR). This year the SNIR moved into space renovated for its use. Major equipment. 1) Zeiss AxioscanZ1 slide scanning microscope. This is a high quality widefield microscope with transmitted brightfield and fluorescent epi-illumination capacity. Its most significant feature is the ability to program multichannel tiled acquisition of large areas from up to 100 microscope slides. It is being actively used by investigators from 10 different intramural laboratories for projects that include whole brain mapping of gene expression profiles and the projections of genetically tagged and fluorescently labeled neuron populations. The system continues to be used to capacity. 2) Zeiss LSM780 microscope is approximately 6 years old. It is a high quality inverted confocal microscope with 405, 488, 514, 561, 594 and 633 nm lasers, a 32-channel GaAsP based spectral detector and 2 conventional PMTs. It is used to capacity during core working hours by investigators from approximately 10 different NIMH intramural groups. 3) A LaVision Ultrascope. This is a light sheet microscope optimized for low magnification (1.2 to 12X 0.5 NA objective with a light sheet thickness minimum of 4 microns) imaging of large samples (up to approximately 10 x 10 x 6 mm). It has 405, 488, 552, 638, and 740 nm lasers. Whole mouse brains immunolabeled with the iDISCO technique are being imaged routinely and projects using brains cleared with CUBIC and other procedures are under development. 4) A Leica SP8 confocal/multiphoton system on an upright microscope frame equipped and with long working distance dipping objectives designed for work with thick cleared samples. It is equipped with 405, 488, 552 and 638 nm fixed lasers and an Insight X3 tunable IR laser and both internal and external non-descanned PMT and HyD detectors. Capacity to perform fluorescent lifetime imaging microscopy (FLIM) was added this year. 5) A Nikon C2 system is now set up for both widefield epillumination and laser scanning confocal imaging. The microscope provides alternatives to the Zeiss Axioscan and LSM780 that are being used to capacity. It also provides the unique capacity to scan large areas in widefield mode at low or moderate magnification and to subsequently image regions of interest at high resolution and in confocal mode. Major Software. Microbrightfield Brainmaker and Neurolucida 360 software packages are available to facilitate reconstruction and analysis of systems neuroscience data. Arivis Vision4D is available for visualization of large 3 dimensional datasets and implementation of analysis pipelines. Training. Practices for in depth training on use of hairpin chain reaction based in situ hybridization and use of each of the microscopes and the software packages described above have been established and implemented for multiple new users. Ongoing assistance with microscope and image analysis software was provided. Several groups have adopted and implemented the whole brain staining and clearing protocols that we provided, while several others have consulted with us and are in early pilot experimental phases of tissue clearing experiments. Publications that used images generated on SNIR microscopes include: Beas BS, Wright BJ, Skirzewski M, Leng Y, Hyun JH, Koita O, Ringelberg N, Kwon HB, Buonanno A, Penzo MA. The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism. Nat Neurosci. 2018 Jul;21(7):963-973. Lehmann ML, Weigel TK, Cooper HA, Elkahloun AG, Kigar SL, Herkenham M (2018) Decoding microglia responses to psychosocial stress reveals blood-brain barrier breakdown that may drive stress susceptibility. Sci Rep 8:11240. Lehmann ML, Weigel TK, Poffenberger C, Herkenham M (2019) The behavioral sequelae of social defeat require microglia and are driven by oxidative stress in mice. J Neurosci. Williams Avram S.K., Lee, H.J., Fastman, J., Smith, A.S., Cymerblit-Sabba A., Vincent, M., Song, J., Granovetter, M.C., Lee, S., Stackmann, M., Chaturvedi, R., Young W. S. NMDA receptor in vasopressin 1b neurons is not required for aggression, short-term social or object memory (June 2019 BiorXiv.org doi: 10.1101/670893)in revision, Frontiers in Behavioral Neuroscience